Supplemental Information Schäfer et al.:

Impaired DNA demethylation of C/EBP sites causes premature aging

Inventory

Supplemental Figures

Figure S1, related to Figure 1

Figure S2, related to Figure 2 and 3

Figure S3, related to Figure 4

Figure S4, related to Figure 5

Figure S5, related to Figure 6

Figure S6, related to Figure 7

Supplemental Tables

Supplemental Table S1, spreadsheet, related to Figure 2

DMR list of whole genome bisulfite sequencing

Supplemental Table S2, spreadsheet, related to Figure 3

GADD45 ChIP-Seq peaks

Supplemental Table S3, spreadsheet, related to Figure 4

Ing1 ChIP-Seq peaks

Supplemental Table S4, spreadsheet, related to Figure 4

RNA-Seq in undifferentiated MEFs

Supplemental Table S5, spreadsheet, related to Figure 4

NG-Capture C viewpoints

Supplemental Table S6, spreadsheet, related to Figure 5

RNA-Seq at day 6 of MEF adipocyte differentiation

Supplemental Table S7, spreadsheet, related to Figure 6

Microarray of white adipose tissue

Supplemental Table S8, related to Figure 7 and S7

Supplemental primer sequences

Supplemental references

2

3

Supplemental Figure S1 | Loss of Gadd45a and Ing1 promotes segmental progeria

(A) Quantification of epidermal and dermal thickness in H&E-stained sections of hind limb skin from Fig. 1D. n=1-4 mice per genotype.

(B) Quantification of senescence-associated β-Galactosidase (SA-βGal) staining in mouse dermis from Fig. 1E; n=5 animals per genotype.

(C) Senescence-associated β-Galactosidase (SA-βGal) assay in MEFs at indicated passages. n=3 independent cell lines per genotype.

(D) Immunoblot of the senescence marker p16INK4ain passage 6 MEFs of indicated genotypes.

-Tubulin served as loading control. n=3 independent MEF lines per genotype

(E) Repair kinetics of T4-endonuclease V-sensitive cyclobutane pyrimidine dimers (CPDs) in passage 4 MEFs was determined at 4h, 8h and 24h after UVB treatment to monitor cellular NER capacity. n= 3 for the 8h data point. At 8h, there was no change in Ing1-/- MEFs vs WT, but a significant change in Gadd45a-/- (p=0.03) and DKO- (p=0.003) vs WT MEFs. The difference between DKOs vs Gadd45a- or vs Ing1 single mutants was statistically not significant.

(F) Fpg (formamidopyrimidine - DNA glycosylase) -sensitive oxidized purines and AP sites, reflecting cellular BER capacity, were determined in passage 4 MEFs in steady-state. n=3

(G) Immunoblot of apoptosis marker Caspase 3 in passage 6 mouse embryonic fibroblasts (MEFs) of indicated genotypes. α-Tubulin was used as loading control. Molecular weight of proteins in kDa is indicated on the left. WT, wildtype; DKO, Gadd45a/Ing1 double knockout

(H) Quantification of γH2A.X staining of WT and DKO MEFs at passages 3 (p3) and 6 (p6) to analyze DNA damage accumulation. n>100 cells per condition and genotype were quantified. Dots indicate numbers of foci in individual cells, red lines indicate median values, whiskers of the box plot represent data within 1.5x interquartile range. Numbers below the box plots indicate average number of γH2A.X foci per cell.

(I) Telomere length in mouse livers of indicated genotype (n=4 per genotype) measured by Southern Blot analysis. RLU, relative length units

(J-K) Quantification of total reactive oxygen species (ROS) (J) and superoxide (K) levels by flow cytometry in passage 3 MEFs (n=4 individual lines per genotype). An N-Acetyl-Cysteine treated negative control (-) and a Pyocyanine-treated positive control (+) served as calibration references.

(L) Gene expression analysis of WNT target genes as an indicator of stem cell function in mouse livers (n=9 per genotype).

(M) Protein levels of senescence-associated secretory phenotype (SASP) related cytokines measured by a cytokine protein array in mouse serum (three pooled serum samples per genotype). Signal intensities are only comparable within analysis of the same protein.

Data of bar charts represent mean values of the indicated number of samples +/- SD.

*, p<0.05; ***, p<0.001. G45a, Gadd45a

4

5

Supplemental Figure S2 | Whole-genome bisulfite sequencing reveals local DNA

hypermethylation in Gadd45a/Ing1 DKO-MEFs

(A) Histogram of distribution of DNA methylation levels measured by Whole-genome bisulfite sequencing (WGBS) in wildtype (WT) and Gadd45a/Ing1 double-knockout (DKO) MEF lines.

(B) Genome-wide correlation of DNA methylation levels at individual CpGs in WT and DKO MEF lines.

(C) Confirmation of hypermethylated DMRs. Methylation-sensitive qPCR measurements of (5mC+5hmC) levels at CpGs within the unmethylated Gapdh promoter, the imprinted Peg3 locus, and several hypermethylated differentially methylated regions (DMRs) in MEFs of the indicated genotypes.

(D) Histogram showing size distribution of hypermethylated DMRs of DKO MEFs.

(E) Genomic locations of DKO hypermethylated DMRs. Numbers indicate absolute numbers of DMRs within each category. DMRs that fall within more than one category were counted in each category. Active promoters are defined as regions ± 1 kb of transcription start site (TSS) decorated by H3K4me3. Enhancers are defined as regions decorated by both H3K4me1 and H3K27ac.

(F) Exemplary DMR at the Kitl gene in WT vs. DKO MEFs. Methylation at individual CpGs as measured by WGBS; MEF histone marks are from (Yue et al. 2014).

(G) GADD45α-ChIP-qPCR. MEFs were transiently transfected with either control or Cebpb- and/or Cebpd- specific siRNA, followed by transient HA-Gadd45a- (HA-G45a) or control- (Ctrl) transfection. GADD45α binding to 12 genomic regions identified in the ChIP-Sequencing was analysed. An unrelated control region served as negative control (Ctrl). Data represent the mean of biological triplicates +/- SD. Cebpb and Cebpd knockdown was controlled by qPCR and greater than 80% or 50%, respectively (data not shown). ns, not significant

 

6

7

Supplemental Figure S3 |  ING1 binds to hypomethylated promoters and can loop to

GADD45α sites

(A) Heatmap of mean differential methylation in 1kb bins around ING1 ChIP-Seq peaks (P). Data are presented as DNA methylation difference between DKO MEFs and WT MEFs as determined by WGBS.

(B-C) Exemplary genomic loci showing GADD45α and ING1 occupancy in WT and DKO MEFs. Methylation at individual CpGs as measured by WGBS, ChIP-Seq profiles of transfected HA-tagged ING1 and GADD45α in MEFs with their respective GFP control from this study; MEF histone marks from (Yue et al. 2014). (B) Example of overlapping GADD45α and ING1 ChIP-Seq peaks around the TSS of the Hipk3 gene. (C) Example of an enhancer-associated GADD45α peak in vicinity of a promoter-associated ING1 peak at the Sec24b gene.

(D) Overlap between Tet1/2 DKO deregulated genes (Wiehle et al. 2015) and ≥ 1.5-fold-downregulated genes in Ing1-/- or DKO MEF.

(E) Genes hypermethylated and downregulated in DKOs MEFs are enriched for C/EBP motifs.

Hypermethylated DMR-associated genes from Ing1/Gadd45a DKO MEFs (n=1331, closest genes determined by analysis with GREAT, http://great.stanford.edu/public/html/) were intersected with genes downregulated in DKO MEFs (n=966 with log2 fold-change < -0.35 at 10% FDR). Overlapping genes were subjected to geneset enrichment analysis of transcription factor targets from the ChIP-X database (ChEA 2016-Enrichr http://amp.pharm.mssm.edu/Enrichr). Enriched transcription factors are displayed on the Y-axis along with PMIDs of the original studies. The combined score represents the enrichment and is calculated by Enrichr from p-value (Fisher exact test) and the z-score of the deviation from the expected rank. Adjusted p-value (q) is indicated in the bar chart. Note that

downregulated genes are highly enriched for C/EBP binding sites.

(F) Top, GADD45α- and ING1-ChIP-seq peaks, hypermethylated DMRs identified in Gadd45a/Ing1 double knockout (DKO) MEFs, and adipogenic hotspots (based on (Siersbaek et al. 2014)). Bottom, NG-Capture C interaction profile of all wildtype (WT), Gadd45a-/- (G45a), Ing1-/- (Ing1) and DKO MEF lines in triplicates in a 1.3Mb genomic region on chromosome 5. The interaction between the GADD45α-bound viewpoint (orange) with the Tgfbr3 promoter bound by ING1 is highlighted (grey shading). Replicate 2 of WT and of DKO are shown in Fig. 4H.

 

8

9

Supplemental Figure S4 | GADD45α and ING1 are required for adipocyte differentiation downstream of C/EBPβ

(A) Quantification of Fig. 5b. Areas with positive staining for Oil Red O were quantified in 9 overview micrographs per MEF line and time point. *, p<0.05 compared to wildtype (WT) at same time point.

(B-C) Transcriptome analysis in differentiating MEFs. RNA-seq analysis at day six of MEF-to-adipcoyte differentiation showing overlap of ≥2-fold downregulated (B) or upregulated (C) genes at 10% FDR in indicated genotypes compared to WT. Values indicate numbers of deregulated genes in the respective genotype.

(D-F) qPCR expression analysis of late adipogenic marker genes Leptin (D), Adiponectin (E) and Fabp4 (F) during MEF-to-adipocyte differentiation in indicated genotypes. Data are presented as mean values of three independent MEF lines per genotype +/- SD.

(G) Immunofluorescence microscopy of endogenous C/EBPβ during MEF-to-adipocyte differentiation in indicated genotypes and time points. Note the translocation to the nucleus at 2h and foci formation at 12h, which are unaltered in DKOs.

(H) Luciferase reporter assay for a C/EBP-responsive reporter transiently overexpressed in WT and DKO cells during MEF-to-adipocyte differentiation. Data are presented as mean values of three independent MEF lines per genotype +/- SD.

(I) Gadd45a or Ing1 was transfected in WT and DKO MEFs and differentiated to adipocytes for four days. Expression of the adipogenic marker genes Pparg and Cebpa was measured by qPCR. Data are represented as mean values of three independent MEF lines +/- SD. p-value shown for most relevant expression changes. *, p<0.05

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11

Supplemental Figure S5 | Synthetic lipodystrophy in Gadd45a/Ing1 DKO mice

(A) In situ overview of mouse abdominal cavities of representative animals for each genotype. L, liver; I, intestines; A, gonadal white adipose tissue (gWAT). The white arrow points to reduced gWAT depots of the Gadd45a/Ing1 double knockout (DKO) mouse. WT, wildtype

(B) Histological image (H&E stain) of interscapular brown adipose tissue (BAT). Representative images from n=3-6 animals per genotype.

(C) qPCR expression analysis of brown adipose tissue (BAT) related genes in gonadal white adipose tissue (gWAT) (n=5-9 mice per genotype).

(D) qPCR expression analysis of brown adipose tissue (BAT) related genes in BAT (n=3-5 mice per genotype).

(E-F) Transcriptome analysis in WAT. Microarray expression analysis was performed on gWAT from three pools of three mice each per genotype. (E) Bar chart showing the number of ≥ 1.5-fold up- and downregulated genes at 10% FDR, respectively, in indicated genotypes compared to WT. No genes were significantly up- or downregulated in Gadd45a-/- mice. (F) Heatmap of ≥ 1.5-fold down- (green) and upregulated (red) genes in DKO gWAT at FDR 10%. Note that most of the expression changes in Ing1-/- single mutants, visible in (F), are not statistically significant (see bar chart in (E)).

12

Supplemental Figure S6 | GADD45/ING1 promote C/EBP function and impact aging

(A-C) Quantification of apoptotic cells per follicle (A), corpora lutea per ovary (B), and follicles per ovary (C) in H&E stained ovary sections of indicated genotypes (Fig. 7E). Data are presented as mean values of n=5 animals per genotype +/- SD.

(D) Overlap of synthetic phenotypes of Gadd45a/Ing1 DKO mice with phenotypes exhibited by mice

mutant for Cebp and progeroid mice, respectively (see also Supplemental Table S8).

13

Supplemental Tables S1-S7 as spreadsheets

Supplemental Table S8: Occurrence of symptoms associated with aging in < 2 months old

Gadd45a-Ing1 double-knockout (DKO) and C/EBP KO mice.

Symptom Occurence in Gadd45a-Ing1 DKO mice

Occurence in C/EBP KO mice

Occurence in prematurely aging mice

Weight reduction yes Rahman et al. 2012; Staiger et al. 2009; Wang et al. 1995

Andressoo et al. 2009; de Boer et al. 1998; Harada et al. 1999; Mounkes et al. 2003; Niedernhofer et al. 2006; Pendas et al. 2002; Trifunovic et al. 2004; van de Ven et al. 2006; van der Pluijm et al. 2007

Lipodystrophy yes Rahman et al. 2012; Staiger et al. 2009; Tanaka et al. 1997; Wang et al. 1995

de Boer et al. 1998; Hemmeryckx et al. 2012; Karakasilioti et al. 2013; Peinado et al. 2011; Pendas et al. 2002; Trifunovic et al. 2004; van der Pluijm et al. 2007

Decreased adipogenic differentiation

yes Tanaka et al. 1997 Compe et al. 2005

Dampened GH-IGF1 axis

yes Arizmendi et al. 1999; Rahman et al. 2012; Staiger et al. 2009

Niedernhofer et al. 2006; van de Ven et al. 2006; van der Pluijm et al. 2007

Hypoglycemia Yes Arizmendi et al. 1999; Liu et al. 1999; Rahman et al. 2012; Wang et al. 1995

Niedernhofer et al. 2006; van de Ven et al. 2006

Ovarian atrophy yes Sterneck et al. 1997 de Boer et al. 2002; Kuro-o et al. 1997

Female infertility yes Sterneck et al. 1997; Tanaka et al. 1997

de Boer et al. 1998; Kuro-o et al. 1997; Trifunovic et al. 2004

Perinatal death (partial penetrance) *

yes Tanaka et al. 1997 van der Pluijm et al. 2007

Reduced life-span yes Screpanti et al. 1995 Andressoo et al. 2009; de Boer et al. 1998; Harada et al. 1999; Kuro-o et al. 1997; Mounkes et al. 2003; Niedernhofer et al. 2006; Trifunovic et al. 2004; van de Ven et al. 2006; van der Pluijm et al. 2007; Vogel et al. 1999

14

Inflammation-signature yes Huggins et al. 2013 Karakasilioti et al. 2013; Lumeng et al. 2011

WAT browning yes Rahman et al. 2012

Reduced gluconeogenesis

yes Arizmendi et al. 1999; Inoue et al. 2004; Liu et al. 1999; Wang et al. 1995

Small body size yes Andressoo et al. 2009; Harada et al. 1999; Kuro-o et al. 1997; McWhir et al. 1993; Mounkes et al. 2003; Niedernhofer et al. 2006; Pendas et al. 2002; van de Ven et al. 2006; van der Pluijm et al. 2007; Vogel et al. 1999

Kyphosis yes Andressoo et al. 2009; de Boer et al. 2002; Kuro-o et al. 1997; Niedernhofer et al. 2006; Pendas et al. 2002; Trifunovic et al. 2004; van de Ven et al. 2006; van der Pluijm et al. 2007; Vogel et al. 1999

Bone marrow fattening yes Moore and Dawson 1990; Prasher et al. 2005

Skin senescence yes Dimri et al. 1995; Kuro-o et al. 1997

Decreased cell proliferation

yes Mounkes et al. 2003; Niedernhofer et al. 2006

Decreased bone density n.d. Smink et al. 2009 Brennan et al. 2014; Chen et al. 2013

Increased apoptosis n.d. Harada et al. 1999

Increased DNA double strand breaks

n.d. White et al. 2015

Telomere shortening n.d. Jaskelioff et al. 2011; Rudolph et al. 1999

Increased ROS production

n.d.

Change in WNT signaling

n.d. Brack et al. 2007; Castilho et al. 2009; Liu et al. 2007

Increased circulating senescence-associated factors

n.d. Coppe et al. 2008; Coppe et al. 2010

Comments:

* Not mentioned in the manuscript n.d. No difference

15

Supplemental Material and Methods

Primer sequences: qRT-PCR primers

Target Forward primer sequence Reverse primer sequence UPL probe Acadl TGGGGACTTGCTCTCAACA GGCCTGTGCAATTGGAGTA 103 Acadm AGTACCCTGTGGAGAAGCTGAT TCAATGTGCTCACGAGCTATG 110 Acadsb TCCAGATAGGGAAACGAGAAAA TCCCCAAAATATTAGTCTCTGGAA 105 Adiponectin GGAGAGAAAGGAGATGCAGGT CTTTCCTGCCAGGGGTTC 17 Aldh2 TGTTCGGGGACGTAAAAGAC TGAGGATTTGCATCACTGGT 63 Cebpa AAACAACGCAACGTGGAGA GCGGTCATTGTCACTGGTC 67 Cebpb TGATGCAATCCGGATCAA CACGTGTGTTGCGTCAGTC 102 Cebpd CTTTTAGGTGGTTGCCGAAG GCAACGAGGAATCAAGTTTCA 33 Cidea TTCAAGGCCGTGTTAAGGA CCTTTGGTGCTAGGCTTGG 46 Cox4i TCACTGCGCTCGTTCTGAT CGATCGAAAGTATGAGGGATG 7 Cox5b CTCCATGGCTTCTGGAGGT GCCTTTGGAGGTAGCATATTGT 26 Cyt c AACGTTCGTGGTGTTGACC TTATGCTTGCCTCCCTTTTC 104 Dhrs4 GAGTGTGACTGGCATCGTGT ATATCAATCCCCTGGTGACG 60 Elovl3 ACTTCGAGACGTTTCAGGACTTA GACGACCACTATGAGAAATGAGC 25 Elovl4 ACGACACCGTGGAGTTCTATC GCGGCCAGTCTGCTACAC 85 Fabp4 AAGAGAAAACGAGATGGTGACAA CTTGTGGAAGTCACGCCTTT 31 G6pc TCTGTCCCGGATCTACCTTG GAAAGTTTCAGCCACAGCAA 19 Gadd45a GCTGCCAGCTGCTCAAC TCGTCGTCTTCGTCAGCA 98 Gapdh AGCTTGTCATCAACGGGAAG TTTGATGTTAGTGGGGTCTCG 9 Idh3b GCTGCGGCATCTCAATCT CCATGTCTCGAGTCCGTACC 67 Igf1 CTGCTTGCTCACCTTCACC AGCCTGTGGGCTTGTTGA 22 Ing1 CCTCCTTCTTCGTGCAGATTG TCTGGCGTTTGAACTTGTCATAG 17 InsR GAGAATTTCCTTCACAATTCCATC CACTTGCATGACGTCTCTCC 104 Irs1 CTATGCCAGCATCAGCTTCC TTGCTGAGGTCATTTAGGTCTTC 71 Leptin CAGGATCAATGACATTTCACACA GCTGGTGAGGACCTGTTGAT 93 Ndufab1 TGCAGATAAGAAGGATGTGTATGAA CTGTCAGTCGGCCACGAT 109 Ndufv1 GGGTGAGATGAAGACATCAGG TCAGCATTCACCACCAGATACT 60 Pepck GGAGTACCCATTGAGGGTATCAT GCTGAGGGCTTCATAGACAAG 49 Pex19 TGCTGTACCCATCCCTGAA GGAGGAGTGGAGTCCTGGT 50 Pex3 CAATGTGGAATTTTCTGAAACG TCTGTCCATATTTTCCCAGGAT 104 Pgc1a GAAAGGGCCAAACAGAGAGA GTAAATCACACGGCGCTCTT 29 Plin1 AACGTGGTAGACACTGTGGTACA TCTCGGAATTCGCTCTCG 64 Ppara CACGCATGTGAAGGCTGTAA GCTCCGATCACACTTGTCG 41 Pparg CAAGCCCTTTACCACAGTTGA CAGGTTCTACTTTGATCGCACTT 67 Prdm16 TCTCGGATCCCATCCTCA GGAAGATCTTGCCACAGTACCT 12 Tbp GGGGAGCTGTGATGTGAAGT CCAGGAAATAATTCTGGCTCA 97 Ucp1 GGCCTCTACGACTCAGTCCA TAAGCCGGCTGAGATCTTGT 34 Ucp3 TACCCAACCTTGGCTAGACG GTCCGAGGAGAGAGCTTGC 69 Axin2 GAGAGTGAGCGGCAGAGC CGGCTGACTCGTTCTCCT 96 Dkk3 GCCTGAAGGAGCTTTGGAC GGCTTGCACATGTACACCAG 76 Dkk4 ACGAAGAAATCACAAAGCAGTAAG AAAAATGGCGAGCACAGC 81 Kremen1 CCCCAGGTAGCCATTCCT CAGGCCGTACACAGTCCA 5 Rspo1 CGACATGAACAAATGCATCA CTCCTGACACTTGGTGCAGA 5 Rspo3 TCAAAGGGAGAGCGAGGA CAGAGGAGGAGCTTGTTTCC 103 Frzb CACCGTCAATCTTTATACCACCT TCAGCTATAGAGCCTTCTACCAAGA 34 Ccnd1 TTTCTTTCCAGAGTCATCAAGTGT TGACTCCAGAAGGGCTTCAA 72 Myc CCTAGTGCTGCATGAGGAGAC TCCACAGACACCACATCAATTT 77

16

ChIP-qPCR primers (Fig. 2I and Fig. 6F)

Note: Primers are not identical with Supplemental Fig. S2C

Primer Genomic coordinates (NCBI37/mm9) Primer sequence (forward and reverse) UPL probe

CTRL1

(Gapdh)

chr6:125115169-125115257

f: CCTACGCAGGTCTTGCTGA 10

r: TGACCTTGAGGTCTCCTTGG

CTRL2

chrX:83550052-83550147

f: CAGTTGGAAAGTCTCAGAGAGTAGAG 48

r: TGTGGTGGTATGGGTGCTC

DMR1

chr4:97771169-97771233

f: CACTGCAGGAGGCCAAAC 107

r: TGGGTCTCCATAGCACAGC

DMR2

chr5:107420642+107420733

f: ACCTTGGTGCACTCCATAAAGA SYBR

r: CACTGCGGTGAGGAGAGAGT

DMR3

chr17:50250575+50250654

f: AACATTCTGCAGCCCATTTC 6

r: ACAACCAGGGCATTGCTTA

DMR4

chr4:80955790+80955906

f: GCAAGAGAGCTGATCCCGTA SYBR

r: TGACATCACCTCCCTCCTTT

DMR5

chr12:76803394+76803467

f: CTGGAGCCGTTTCTTCAGG 62

r: ATTTTGTGGTGGGCTTCCTA

DMR6

chr1:170292711-170292778

f: GGGACAGGGAGCTACTAGGAA 67

r: TCGCACTTGGCAAGACTCTA

DMR7

chr5:107420675-107420749

f: GATAATCCAAACAAAGCTAAGAATGTT 91

r: AGTTGGATTCATCTCACACTGC

DMR8

chr4:6149607-6149696

f: TGGAAAGTTTTCAACCTGCTG SYBR

r: TACAAACTTATTTCAGACTACATGACC

DMR9

chr7:141369180+141369299

f: CTTGTTGACAAACTGTAAAAGGAG SYBR

r: TGTCGATGCCCAAATACAGA

no DMR

chr5:106049748+106049818

f: CAATACCACAGTAAGAGCTGCCTAA 19

r: GGAAGCCCTGAGAGAGCTG

17

ChIP-qPCR primers (Supplemental Fig. S2G)

ChIP Primer Genomic coordinates (NCBI37/mm9) Primer sequence UPL probe

CTRL1 (Gapdh) chr6:125115169-125115257 f: CCTACGCAGGTCTTGCTGA

10 r: TGACCTTGAGGTCTCCTTGG

1 chr13:90028893-90028952 f: GCCTAGCTGCAGTTTCATCA

66 r: CTGACTCCACTGAGGCTGATT

2 (=2I, DMR3) chr17:50250575-50250654 f: AACATTCTGCAGCCCATTTC

6 r: ACAACCAGGGCATTGCTTA

3 chr12:76803394-76803467 f: CTGGAGCCGTTTCTTCAGG

62 r: ATTTTGTGGTGGGCTTCCTA

4 chr12:76803468-76803557 f: TCCGTATCTCCCTCCTAACACT

100 r: CGTGTGTTAGACAATCTAGACGTTG

5 chr7:138086947-138087019 f: GTTGCGCAATGAGCTCTCTA

27 r: CACGCTGAGACAAACACACTC

6 chr11:50192068-50192161 f: CCGCTGTTACGCAATGGT

89 r: AGTTTTAAAAGAAAACAAATTCAGCAG

7 chr8:122364548-122364618 f: ATCCCAGTCTGTGCGGATT

19 r: GAAGGGAAGACGAGGAGGAA

8 chr6:115403104-115403165 f: AGCCCAGTGCAGAGTTTAGC

48 r: GGTTAGATTTTTGGGAGGAAGG

9 (=2I, DMR6) chr1:170292711-170292778 f: GGGACAGGGAGCTACTAGGAA

67 r: TCGCACTTGGCAAGACTCTA

10 chr4:97770997-97771069 f: TGGTTTGGACAAGTGCTGTG

29 r: CAGGAGCATGTGCAATAGTTTT

11 chr4:57852232+57852354 f: AAATTAAGTTCTGTGACTTTTGACTCC

17 r: GGGGCTGTCCGTAGACATT

12 (=2I, DMR7) chr5:107420675-107420749 f: AAATTAAGTTCTGTGACTTTTGACTCC

91 r: AGTTGGATTCATCTCACACTGC

18

MS-qPCR primers (Supplemental Fig. S2C)

Note: Primers are not identical with Fig. 2I

Primer Genomic coordinates (NCBI37/mm9) Primer sequence (forward and reverse) UPL probe

Gapdh chr6:125115169-125115257 f: CCTACGCAGGTCTTGCTGA 10

r: TGACCTTGAGGTCTCCTTGG

Peg3 chr7:6683069-6683210 f: AGTGGACCCACACTGAACC 26

r: GAGAAGCGGAGAGATGTCCA

DMR1 chr2:173091915-173092050 f: TTCCTCCTATGGCTGTGACC 33

r: AAGGCGTGAATCATTTATTGGT

DMR2 chr18:23940746-23940854 f: TTCTTTGTTTAACTTGTCATCTAGCC 9

r: GGTTGGGAGACATAAGGACTCA

DMR3 chr18:23940753-23940854 f: TTTAACTTGTCATCTAGCCTCAGC 9

r: GGTTGGGAGACATAAGGACTCA

DMR4 chr4:105314322-105314417 f: GTTCACTTCTTGAAGCTTTTCTCA 55

r: CAACATGTTTATTCACATGGAGAGA

DMR5 chr16:92449531-92449606 f: TGCGGTTAAAAGTCTCATGC -

r: ATTCGGAGCGTGTTGTTCTT

DMR6 chr8:49394994-49395067 f: GGAAACCCAAGAGGGGAAA -

r: TCAGTGAAAAGATGTGTTCGAGA

DMR7 chr6:4460009-4460109 f: TGGAAATAATTGTGAAGCAGTCA -

r: CACCGAAATGTAGCAAAAATG

DMR8 chr2:118844243-118844321 f: CGATGAGTGTGTGAGCGAAT -

r: AGGAACAAGGGTAGCCAAGG

DMR9 chrX:166435711-166435776 f: CCCTAGGTAGCGCAGTGAGT -

r: ACACGGGGTCTCTGTGTCTG

DMR10 chr7:13489136-13489248 f: GGAATCCAGCCCTAGCTTTAC 42

r: GCTCTCGCAGTTGGGCTA

DMR11 chr11:49624657-49624751 f: TCCCTAGTGTGAGCCTTAGCC 102

r: CACAGCAGCCCTCAAGATAA

DMR12 chr19: 38746596-38746995 f: GCAATCCTAACAAAAATACTGAAGTG 97

r: GGCTCTGACACTGTGGTTGA

DMR13 chr18:66287008-66287118 f: AAAGCTATGCCTTGGAACCAT -

r: TTCCCATTCAGGGCTGTAAA

DMR14 chr5:53794142-53794231 f: TGAGTGTGCTATGTGTCAGCAG 6

r: AGGAAGCTAGTTCTACGAATCCAC

DMR15 chr10:99495588-99495662 f: CATCCCTTCCAAACGTGTCT -

r: TCATGATGACTCCACACAGGA

DMR16 chr18:23940654-23940729 f: TGGTGGCTCACCCAGAGT 66

r: GGCTGGTCTCTTGGGACA

DMR17 chrX:166435106-166435188 f: ACACAGCGTGAACGACCAG -

r: GCCTGAGACGAATCAATGAAA

DMR18 chr13:113545830-113545918 f: ACCACGGAGCTGAGTCCTAA 53

r: AGGAATCAGGAGACAACTATGGA

19

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